Run Scaffold 5.3.3 and choose new analysis.
Select Quantitative Technique: Check TMTpro 16plex -->next
New TMT-16plex Sample --> Check MudPit Experiment (to combine the 8 fractions (8 MSF files) into one analysis output) --> next
Queue Search Engine for Landing --> select all 8 MSF files and add to Import Queue.
Load and Analyze data --> Enter the database which was used in PD for the search. You will have to index in Scaffold just as you did in PD before you can use a database. Use Legacy LFDR, protein cluster analysis, and pre-compute FDR.
Click 'Load Data' and allow to run.
Once all data is loaded, apply filters using a FDR, Peptide/Protein Prophet or XCorr - or some combination. For our general settings in proteomics we use - min peptide 1, protein prophet 90%, peptide prophet 60%, DeltaCN 0, Xcorr - +1>1.8, +2>2.0, +3>2.2, +4>3.0.
At the sample view, by default it displays one sample column and quantitative value for each identified proteins, in order to get the TMT tag intensities, enter sample grouping and apply statistics do the following.
Click on Q bottom: Launch Q+ Quantitation (iTRAQ, SILAC, TMT, Label-free ) Module in a new window.
Analysis type: intensity-based --> next
Experiment type: Between-subjects (Independent groups). --> next
Edit sample names and categories --> edit sample name of each tag and for treatment groups --> next
Organize Quant samples --> drag and drop each sample to its group --> next
Settings --> use non-exclusive peptides: false, Calculation type: Median, Blocking level: Unique peptides, Use protein average as reference: True, Spectrum Quality Filter: no filter. --> Finish
Once data is loaded, it displays all tags with sample names as edited and log2 fold changes for each quantified protein.
Quantitative analysis --> apply specific statistics in accordance with the study design and generate graphics and GO to be exported or export data to excel and apply statistics with algorithm of choice to detect disregulated proteins and enriched pathways.